It is our intent to address the specific biochemical mechanisms which give rise to the cytotoxic action of antifolates in tumor cells. Methotrexate is one of the most widely used chemotherapeutic agents for the treatment of many cancers and even though a large number of pharmacologic investigations have been conducted with this drug it is still not completely understood at the biochemical level; a. why this type of drug is so uniquely effective, b. what specific biochemical parameters can be used to judge the effectiveness of the drug and c. the basis for possible selective rescue of normal tissue by leucovorin after methotrexate treatment. To address these questions requires that the target metabolites, reduced folates, be evaluated with sufficient accuracy and sensitivity to permit experimentation on tumor model systems. During previous studies analytical methods were developed for estimation of reduced folates based on incorporation of tritiated fluorodeoxyuridylate into a stable ternary complex with purified thymidylate synthase and tissue extract methylenetetrahydrofolate. The high specificity and stability of this complex permits accurate estimation of subpicomole quantities of tissue extract methylenetetrahydrofolate. Dihydrofolate, tetrahydrofolate, 5-methyltetrahydrofolate and 10-formyltetrahydrofolate can also be determined after chemical or enzymatic cycling to methylenetetrahydrofolate. This methodology will be used to investigate changes in these metabolites in murine leukemia L1210 cells and mouse small intestine epithelial cells in addition to rat hepatoma cells and isolated hepatocytes. The approach will involve comparison of normal vs. tumor tissue with regard to the sensitivity of reduced folates to methotrexate and other antifolates, evaluation of leucovorin rescue from high dose methotrexate, impact of inhibitors of folylpolyglutamate synthetase on intracellular folypolyglutamates, and evaluation of polyglutamyl hydrolase activation by methotrexate. These studies will provide a better understanding of how this important class of cancer chemotherapeutic agents can be used to selectively destroy cancer cells while minimizing damage to host tissue.